doing phage titrering

mountain

hi guys!

i am doing phage titrering but have difficulty in chosen the right values. please give me some ideas. thanks in advance!


here is what we did:
dilute phages 5X with dilution buffer. add 1ul of the diluted phage to 200ul of the XL1-Blue overnight culture. Add 2ml melted top agar. Quickly pour onto a plate. the day after count pfu/ml.

pfu/ml: (number of plaque*dilution factor*10^3ul/ml )/ul of diluted phage plated.

my problem is: what is the dilution factor? is that factor 5 or should i also take 200ul of the XL1-Blue cells and 2ml melted top agar in consideration? (practically it seemed like the phages is diluted more than 5X.)

what is the "ul of diluted phage plated"? is that the 1ul diluted phage i added to the other volumes or is that the total volume (1ul phage+200ul XL1-Blue cells+2ml top agar)?


thanks!

iansmith

5X is your dilution factor and does not include the 200 uL of culture and 2 mL of agar. Also, the 200 uL and 2 mL will not dilute your sample since your plating the whole volume and not just part of it. Therefore your are plating all the phage that are in your 1 uL sample.

pfu means plaque forming unit, so you need to use the volume of phage that you used (1 uL) not the total volume. You looking at counting the number of phage in your stock solution.

mountain

thanks!